Methods of using selective chemotherapeutic agents for targeting tumor cells

ABSTRACT

A method for treating cancer tumors, particularly ovarian cancer tumors, is described, where fused cyclic pyrimidine having a cancer treating ability is selectively delivered to an FR expressing cancerous tumor.

CROSS-REFERENCE TO RELATED APPLICATIONS

This utility patent application is a divisional patent application ofand claims the benefit of priority of co-pending prior U.S. patentapplication Ser. No. 11/820,872, filed Jun. 21, 2007, which claims thebenefit of U.S. Provisional Patent Application Ser. No. 60/816,931 filedJun. 28, 2006 (now expired), the disclosure of each of which areincorporated herein by reference into this divisional utility patentapplication.

GOVERNMENT CONTRACT

This invention was supported in part by the National Institutes ofHealth, U.S. Department of Health and Human Services under Contract No.CA 89300. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for providing selectedchemotherapeutic agents which selectively target folate receptors (FR)of cancerous tumor cells and inhibit GARFTase contained in the cells,particularly types of ovarian cancer cells. Specifically, the presentinvention relates a method for the use of fused cyclic pyrroloderivatives, particularly fused cyclic pyrimidines having a long chainCH₂ group between cyclic groups, which themselves selectively targetfolate receptors (“FR”), particularly FR-alpha of cancerous tumor cells.They also inhibit glycinamide ribonucleotide formyltransferace enzyme(GARFTase) in tumor cells, where the fused cyclic pyrimidines themselvesare effective to selectively penetrate inside of the cancerous tumorcells.

2. Description of the Prior Art

Cancer chemotherapy agents as taught, for example in U.S. Pat. No.5,939,420 (Gangjee), do not specifically selectively target cancer tumorcells but target both normal and tumor cells. This lack of selectivityfor tumor cells results in cytotoxicity to the normal cells and is alsoone of the major causes of chemotherapeutic failure in the treatment ofcancer. Further, advanced stage and platinum resistant tumors may bedifficult to treat with traditional chemotherapeutic agents such as, butnot limited to, carboplatin or paclitaxel (docitaxel). Other documentsin this area include J. Med. Chem. 48 (16), 5329-5336, web release dateJul. 9, 2005 “Synthesis of Classical Four-Carbon Bridged 5-SubstitutedFuro-[2-3-d]-pyrimidine and 6-Substituted Pyrrolo[2,3-d]-pyrimidineAnalogues as Antifolates” by A. Gangjee et al.

As is known in the prior art, a type of folate receptor FR, FR-alpha, isoverexpressed on a substantial amount of certain surfaces of a number ofcancerous tumors including, but not limited to, ovarian, endometrial,kidney, lung, mesothelioma, breast, and brain tumors.

In most normal tissues, the FR-alpha is not present and the folic acidis not taken up by the normal cells by way of a reduced folate carriersystem (RFC), thereby leading to selective uptake by tissues such asFR-alpha expressing ovarian tumors. In light of the specificity of folicacid, conjugates of folic acid have been used in the prior art toselectively deliver toxins, liposomes, imaging and cytotoxic agents toFR-alpha expressing tumors.

However, one of the major limitations of the foregoing, such ascytotoxic-folic acid conjugates, is that its use requires cleavage fromthe folic acid moiety to release the cytotoxic drug. Moreover, prematurerelease of the cytotoxic agent during the transport before reaching thetumor destroys selectivity and thereby leads to undesired toxicity innormal cells. This is a very serious detriment scientifically andcommercially.

Further, if the folic acid moiety of the cytotoxic-folic acid conjugateis difficult to cleave, then the anti-tumor activity is hindered as aresult of the inability or reduced ability to release the cytotoxicagent. Accordingly, treatment of the tumor cells with the cytotoxicagent is either hindered or rendered nil as a result of the difficultyin cleaving the cytotoxic agent moiety from the folic acid-basedconjugate.

In spite of the foregoing prior art, however, there remains a very realneed for methods and compositions that selectively target FR-alpha oftumor cells.

An object of this invention is to provide methods for selectivelytargeting FR, particularly FR-alpha, of tumor cells with acancer-treating agent, targeting the GARFTase enzyme.

In a related object, the method does not use conjugated compositions anddoes not need cleavage to release a cytotoxic drug.

In yet another related object, the method will allow penetration intothe cancerous cells, expressing FR, that is, FR-alpha and/or FR-beta,but not into a cell using the reduced folate carrier system (RFC).

Another object of this invention is to provide effective delivery of acancer treating agent to the cancerous tumor in the process of treatinga patient.

Another object of this invention is to efficiently target a canceroustumor.

Another object of this invention is to provide an essentially non-toxicmethod of treating a cancerous tumor.

SUMMARY OF THE INVENTION

The present invention has filled the above described need and satisfiedthe above objects by providing methods for selectively targeting FR oftumor cells with a cancer-treating agent. The term “FR” as used hereinincludes receptors selected from the group consisting of FR-alpha,FR-beta and mixtures thereof. Folate receptors of the FR-beta-type areexpressed on surfaces of myeloid leukemia cancerous tumors. In apreferred embodiment, the compositions and methods selectively targetFR-alpha and FR-beta of cancerous tumor cells.

Very significantly, in the method, the cancer treating agent is notsignificantly taken up by a cell or tissue using the RFC system.

The cancer treating agent used in this method is a fused cyclicpyrimidine and is used to selectively target FR of ovarian tumors,advanced stage cancerous tumors that express FR receptors anddrug-resistant tumors such as, but not limited to, those resistant tocarboplatin, paclitaxel, and/or docitaxel. The receptors are preferablyFR-alpha and beta types.

More specifically, the invention relates to a method of inhibitingGARFTase in a cancerous tumor of a patient comprising:

(a) providing a fused cyclic pyrimidine shown in FIGS. 1( a) and (b),where n=3-6 alkyl chain carbons between the major ring groups I and II;

(b) selectively delivering the fused cyclic pyrimidine alone to a FRcancerous tumor, where due to the use of long chain carbons (n=3-6), thefused cyclic pyrimidine targets primarily cancerous tumors which containFR. The fused cyclic pyrimidine enters said cancerous tumors, acting asa cancer-treating agent itself, and inhibits GARFTase within the tumors.Here the fused cyclic pyrimidine functions as a substrate offolylpolyglutamate synthetase (FPGS) in the tumors therefore beingtrapped in the tumors.

The distance and orientation of the side chainp-aminobenzoyl-L-glutamate moiety with respect to the pyrimide ring areextremely important for biological activity; hence, n=3-6 in FIGS. 1( a)and (b) provide surprisingly unique results. Here the fused cyclicpyrimidine acts as carrier, targeting and cancer treating agent. Nofusing of a separate cancer treating agent to the fused cyclicpyrimidine is required.

The invention will be more fully understood by review of the drawings inview of the following detailed description of the invention, and theclaims appended thereto.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1( a) shows a general chemical formula for the fused cyclicpyrimide used in the method of this invention, where “L-Glu” is aL-Glutamic Acid (or L-Glutamate) group based on an amino acid having theformula C₅H₉—NH₄; and

FIG. 1( b) shows another description of the formula of FIG. 1( a), wheren is the total number of CH₂ groups between the major cyclic/ringgroups, such groups shown as I and II.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

As used herein, “tumor” refers to an abnormal growth of cells or tissuesof the malignant type, unless otherwise specifically indicated and doesnot include a benign type tissue. The “tumor” may be comprised of atleast one cell and/or tissue. The term “inhibits or inhibiting” as usedherein means reducing growth or replication. As used herein, the term“cancer” refers to any type of cancer, including ovarian cancer,leukemia, lung cancer, colon cancer, CNS cancer, melanoma, renal cancer,prostate cancer, breast cancer, and the like. As used herein, the term“patient” refers to members of the animal kingdom including but notlimited to human beings. The fused cyclic pyrimidine of the inventionhas five unique properties: 1) inhibition of FR-alpha and beta canceroustumors; 2) a lack of appreciable uptake by the RFC; 3) ability to actitself as a cancer treating agent; 4) ability to penetrate canceroustumors having folate receptors; 5) ability to function as substrate forfolylpolyglutamate synthetase (FPGS) thereby being trapped in the tumorcells and 6) inhibition of GARFTase. The fused cyclic pyrimidine of thisinvention targets cancers with certain receptors and is practicallynon-toxic. These fused cyclic pyrimidines are taken into the tumorcells, via the FR selectivity.

Selectivity of the fused cyclic pyrimidine is made possible since mostnormal cells do not have receptors of the FR type. Further, whenexpressed, FR-alpha is the most widely expressed receptor isoform inadult tissue. FR-alpha occurs at the apical (i.e., luminal) surface ofepithelial cells where it is not supplied by folate in the circulation.

Embodiments of the invention follow. The fused cyclic pyrimidine wheren=3-6 has an affinity for receptors such as FR or FR-alpha. Thesereceptors are mainly present on the surface of cancerous tumor cells,and not other types of folate transport systems that are morepredominant on the surface of normal cells. In other words, the fusedcyclic pyrimidine is preferably not taken up to an appreciable degree bythe reduce folate carrier (RFC) system. FR-alpha are generally notexpressed in normal cells. The fused cyclic pyrimidine stays inside ofthe cancerous tumor cell for an effective amount of time to kill thetumor cell. This occurs by way of polyglutamylation and the multi ionicform of the fused cyclic pyrimidine itself inside of the tumor cell. Thefused cyclic pyrimidine also disrupts the replication process of thecancerous tumor cell, thereby inhibiting the growth of FR-alphaexpressing cancerous tumor cells.

The foregoing embodiments are enabled by way of a glycinamideribonucleotide formyltransferase (“GARFTase”) inhibition. GARFTase is anenzyme which is essential to DNA synthesis of normal and cancerous tumorcells.

Here the fused cyclic pyrimidine itself has a high affinity for theFR-alpha receptors which are overexpressed on the surface of canceroustumor cells. The fused cyclic pyrimidine passing into the canceroustumor cells inhibits GARFTase activity and inhibits DNA synthesis.Accordingly, the targeted tumor cells which overexpress FR-alpha areprevented from replicating and are killed.

In a preferred embodiment, the fused cyclic pyrimidine has asignificantly greater affinity for FR-alpha expressing cells comparedwith cells that do not express FR-alpha. Accordingly, the fused cyclicpyrimidine would have a greater affinity for cells which overexpressFR-alpha (i.e., certain cancerous tumor cells as described in moredetail above), but also an affinity for FR-beta cells.

At present, there are no other agents known with the above-describedfour attributes in a single chemotherapy agent and therefore thepresently invented compositions are felt to be unique with regard toother GARFTase or FR-alpha targeting agents, including any known agentin clinical or investigational use.

EXAMPLES

This example relates to just one of the many possible targets, ovariancancer.

Materials and Methods

Pyrrolo derivatives (fused cyclic pyrimidines), such as shown in FIGS.1( a) and (b) (n=3 or 4) AAG366 and AAG344 respectively. These compoundswere designed using molecular modeling, superimposition and/or dockingonto an X-ray crystal structure of GARFTase while maintaining FR-alphatargeting ability using structure based design.

Testing.

-   -   AAG366 and AAG344 were evaluated in two assays, in vitro and in        vivo. The following four properties were considered:    -   Single digit nanomolar inhibition of FR-alpha expressing tumor        cells. Substrate for FPGS (attributes of AAG366 FIG. 1, where        n=3 and AAG344 where n=4).    -   Method of selectively delivering a cytotoxic GARFTase inhibitor        to tumor cells. Greater number of FR-alpha expressed on advanced        and platinum resistant tumors.    -   Method of selectively increasing the cytotoxic agent        concentration in the tumors, affording selective delivery of        AAG366 FIG. 1.    -   Method of inhibiting advanced stage and resistant tumors without        major toxicity.

Recently discovered compounds AAG366 (n=3) and AAG344 (n=4), FIGS. 1( a)and 1(b), potently inhibit the growth of FR-alpha expressing KB tumorcells with IC₅₀=2.5 and 2.9 nM, respectively. In addition, thesecompounds are nanomolar inhibitors of glycinamide ribonucleotideformyltransferase (GARFTase), critical enzyme in purine nucleotidebiosynthesis, and are not taken up by the RFC. Both these compounds areremarkably selective for tumor cells expressing FR-alpha. AAG344 isgreater than 345-fold more inhibitory to tumor cells expressing theFR-alpha compared to cells that do not. AAG366 (n=3) is 100-180 foldmore inhibitory.

AAG366, AAG344, the clinically used antifolates methotrexate,pemetrexed, ratitrexed, as well as the most important GARFTase inhibitorDDATHF (Lometrexol) were evaluated. All of the aforementioned compoundswere less potent against FR-alpha expressing RT16 cells compared withAAG366 and AAG344 and none showed selectivity for the FR-alphaexpressing RT16 cells over RFC expressing pc43-10 cells. Unlike AAG366and AAG344, all of these analogs inhibited the RFC expressing pc43-10cells with greater potency (methotrexate, pemetrexed, ratitrexed) orequal potency (DDATHF) compared to the FR-alpha expressing RT 16 cells(AAG366) or KB cells (AAG344) and were thus devoid of selectivity.

Research indicates that AAG366 and AAG344 are the only known compoundswith these three attributes, i.e., 1) GARFTase inhibition; 2) singledigit nanomolar FR-alpha expressing tumor cell inhibition; and 3) a 100to >345-fold selectivity for FR-alpha expressing cells over cells thatlack the FR-alpha, including those with the RFC. These three importantattributes make AAG366 and AAG344 particularly attractive as antitumoragents. Such novel agents could be used alone or in combination withother chemotherapeutic agents to afford synergistic effects againsttumors with reduced toxicity and thus address the major obstacle oftoxicity in cancer chemotherapy.

Impact, for Example with Ovarian Tumors:

The compound indicated in FIGS. 1( a) and (b) (n=3 or 4) specificallytargets the FR-alpha and is not taken up by the RFC in normal cells.Since 90-95% of ovarian cancers overexpress the FR-alpha, one wouldexpect that these compounds will be highly selective for ovarian canceras well as ovarian cancers resistant to one or both of the most widelyused chemotherapeutic agents, carboplatin or paclitaxel (docitaxel). Themechanism of action of the aforementioned agents is different from theGARFTase inhibitors of this invention and selectivity, if any of theseprior reagents, is not based on FR-alpha. The use of FR-alpha targetingGARFTase agents could radically impact the course of treatment outcomesfor ovarian cancer because of their selectivity for FR-alpha expressingovarian tumor cells that could result in “cures” without toxicity tohost cells. Metastasized ovarian cancer cells in the peritoneal cavityalso over express FR-alpha and would be selectively targeted by ouranalogs.

Data has shown that compounds of FIGS. 1( a) and (b) (n=4) at highextracellular levels is able to diffuse through plasma membranes, andhad increased inhibitory potency against CCRF-CEM cell growth in culturecompared to compounds where n=2.

What is claimed is:
 1. A method for inhibiting GARFTase in canceroustumors of a patient comprising: a) providing a fused cyclic pyrimidinehaving the chemical formula:

where n=5 or 6; b) selectively delivering the fused cyclic primidinealone to cervical cancerous tumors, where the fused cyclic pyrimidinedue to the use of CH₂ chains, where n=5 or 6, targets primarilyFR-expressing cancerous tumors; and c) passing of the fused cyclicpyrimidine into said cancerous tumors where the fused cyclic pyrimidineitself acts as a cancer treating agent and inhibits GARFTase within thetumors.
 2. The method of claim 1, wherein said fused cyclic pyrimidineis selective for receptors selected from the group consisting ofFR-alpha, FR-beta and mixtures thereof, associated with expressingcancerous tumors.
 3. The method of claim 1, wherein said fused cyclicpyrimidine is selective for FR-alpha expressing cancerous cells.
 4. Themethod of claim 1, wherein said fused cyclic pyrimidine is notsignificantly taken up by a tissue or a cell using the reduced folatecarrier system (RFC system).
 5. The method of claim 1, wherein the fusedcyclic pyrimidine functions as a substrate of folylpolyglutamatesynthetase (FPGS) in the tumors, thereby being trapped in the tumors. 6.The method of claim 1, wherein the fused cyclic pyrimidine stays insidethe cancerous tumor for an effective amount of time to kill the tumorsby way of polyglutamylation and the multi ionic form of the fusedpyrimidine itself.
 7. The method of claim 1, wherein said fused cyclicpyrimidine requires no separate cancer treating agent or conjugation toa separate cytotoxic agent.
 8. The method of claim 1, wherein said fusedcyclic pyrimidine targets at least one advanced stage cancerous tumor.9. The method of claim 1, wherein said fused cyclic pyrimidine targetsat least one platinum resistant cancerous tumor.
 10. The method of claim1, wherein said fused cyclic pyrimidine targets at least one carboplatinresistant cancerous tumor.
 11. The method of claim 1, wherein said fusedcyclic pyrimidine targets at least one paclitaxel resistant canceroustumor.
 12. The method of claim 1, wherein said fused cyclic pyrimidinetargets at least one docetaxel resistant cancerous tumor.
 13. The methodof claim 1, wherein said fused cyclic pyrimidine is polyglutamylated byfolypoly-gamma glutamate synthetase.
 14. The method of claim 1, whereinsaid fused cyclic pyrimidine is tolerable in vivo.
 15. The method ofclaim 1, wherein n=5 in the chemical formula.
 16. The method of claim 1,wherein n=6 in the chemical formula.
 17. A method for inhibitingGARFTase in cancerous tumors comprising: a) providing a fused cyclicpyrimidine having a cytotoxic capability having the chemical formula:

where n=5 or 6; b) selectively targeting a folate receptor alpha(FR-alpha) expressing cervical cancerous tumor, with said fused cyclicpyrimidine, wherein said cancerous tumor expresses a plurality of FRreceptors; c) selectively delivering said fused cyclic pyrimidine tosaid cancerous tumor; d) passing of the fused cyclic pyrimidine intosaid cancerous tumor; e) retaining said fused cyclic pyrimidine in saidcancerous tumor for a sufficient time for lysing of said canceroustumor; and f) lysing of said cancerous tumor by said fused cyclicpyrimidine binding with said GARFTase to inhibit DNA replication of saidcancerous tumor.
 18. The method of claim 17, wherein said fused cyclicpyrimidine is selective for receptors selected from the group consistingof FR-alpha, FR-beta and mixtures thereof, associated with expressingcancerous tumors.
 19. The method of claim 17, wherein the fused cyclicpyrimidine stays inside the cancerous tumor for an effective amount oftime to kill the tumor by way of polyglutamylation and the multi ionicform of the fused pyrimidine itself.
 20. The method of claim 17, whereinsaid fused cyclic pyrimidine requires no separate cancer treating agentor conjugation to a separate cytotoxic agent.
 21. The method of claim17, wherein said fused cyclic pyrimidine is tolerable in vivo.
 22. Themethod of claim 17, wherein the fused cyclic pyrimidine targetsprimarily FR-alpha expressing cancerous tumors.
 23. The method of claim22, wherein said fused cyclic pyrimidine requires no separate cancertreating agent or conjugation to a separate cytotoxic agent.
 24. Themethod of claim 17, wherein n=5 in the chemical formula.
 25. The methodof claim 17, wherein n=6 in the chemical formula.